Journal: bioRxiv
Article Title: Statins and genetic inhibition of the mevalonate pathway activate an ATF3-STMN2 regenerative program
doi: 10.64898/2026.02.23.707492
Figure Lengend Snippet: (A) Luciferase assay for STMN2 in WT SH-SY5Y cells treated with siRNA against HMGCR and control. Points represent mean of three technical replicates. Unpaired t-test. (B) Pooled CRISPRi-single-cell RNA-seq data from Tian et al (2019) demonstrates that inhibition of the GGTase I beta-subunit ( PGGT1B) promotes STMN2 expression in iPSCs. Asterisks indicate comparison to non-targeting. (C) RT-qPCR for PGGT1B and STMN2 transcripts in WT SH-SY5Y cells treated with ASOs targeting PGGT1B . Points represent average of technical replicates from independent biological replicates. Unpaired t-tests. (D) RT-qPCR for RHOB and STMN2 transcripts in WT SH-SY5Y cells treated with ASOs targeting RHOB . Points represent average of technical replicates from independent experiments. Unpaired t-tests. (E) Experimental scheme for CRISPRi of targets using NGN2-dCas9 cortical neurons. iPSC were transduced using lentiviral constructs bearing dual-guide RNA at day -3, selected using blasticidin, and NGN2 expression was induced through the addition of doxycycline. Cells were harvested at day 11 post-induction for RT-qPCR. (F) Immunofluorescence imaging of NGN2 -dCas9-BFP iPSC neurons. dCas9 is localized to the nucleus and used to repress indicated genes after lentiviral-mediated expression of dual gRNA. (G) CRISPR inhibition of MVD and GGPS1 in iPSC-derived cortical neurons results in a knockdown efficiency of 60% and 40% respectively via RT-qPCR. Points represent average of technical replicates from independent experiments. Unpaired t-tests. (H) RT-qPCR for STMN2 upon CRISPR inhibition of MVD or GGPS1 in iPSC-derived cortical neurons. Points represent average of technical replicates from independent experiments. One-way ANOVA with Dunnett’s multiple comparisons. (I) RT-qPCR of selected targets in day 32 iPSC-derived cortical neurons following 10 days of treatment with ASOs targeting TARDBP, PGGT1B , GGPS1 or MVD . Points represent independent biological replicates. Unpaired t-tests. (J) RT-qPCR for STMN2 expression following ASO knockdowns in (I) . Points represent independent biological replicates. One-way ANOVA with Dunnett’s multiple comparisons. (K) Immunofluorescent staining using STMN2 (red) and TUJ1 (green) antibodies in non-differentiated WT SH-SY5Y cells demonstrate an upregulation in STMN2 expression, including at the nascent growth cone (arrow). Right panels are insets as indicated. Scale bar = 25μm. (L) Immunofluorescent staining of WT SH-SY5Y using TUJ1 antibody (green) indicates neurite length extension following statin treatment. Scale bars = 25μm. (M) GFP overexpression in TDP-43 mutant SH-SY5Y also reveals neurite extension following 1μM cerivastatin treatment. (N) Automated image analysis of retinoic acid differentiated WT-GFP SH-SY5Y demonstrates dose-response increase in neurite length upon treatment with lipophilic cerivastatin and simvastatin, but not hydrophilic pravastatin. Points represent average of technical replicates from independent experiments. One-way ANOVA with Dunnett’s multiple comparisons. (O) Image analysis of TUJ1 staining to measure neurite length and branching in NGN2 iPSC-derived cortical neurons after statin treatment. Unpaired t-tests. Points represent average values from nine images across each well, across two independent experiments. (P) Automated quantification of neurite length in iPSC-derived motor neurons treated with cerivastatin and immunostained with a TUJ1 antibody. Points represent average of measurements from individual wells. One-way ANOVA with Dunnett’s multiple comparisons.
Article Snippet: Induction was performed as hNIL but without the addition of compound E. Long-term NGN2 culture media was comprised of Neurobasal medium (Gibco, 21103-049) + 1% N2 supplement (Gibco, 17502-048), 2% B27 supplement (Gibco, #17504-044), 1% GlutaMax (Gibco, 35050061), 1ng/mL BDNF (Life Technologies, PHC7074), 1ng/ml NT3 (Peprotech, #450-03), 50ng/ml Aphidicolin (CellSignaling, #32774) and 2μg/mL doxycycline (Sigma-Aldrich, D5207).
Techniques: Luciferase, Control, Single Cell, RNA Sequencing, Inhibition, Expressing, Comparison, Quantitative RT-PCR, Construct, Immunofluorescence, Imaging, CRISPR, Derivative Assay, Knockdown, Staining, Over Expression, Mutagenesis
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